Guinea Pig Liver Mu-Class GlutathioneS-Transferase M1-2 Cross-Reacts with Antibodies to both Rat Mu- and Theta-Class GlutathioneS-Transferases

Two novel major heterodimeric Mu-class glutathione (GSH)S-transferases (GSTs), designated M1-2 and M1-3*, were isolated from guinea pig (gp) liver cytosol and purified to homogeneity together with a known majorhomodimeric Mu-class gpGSTM1-1 (reported as GST b by R. Oshino, K. Kamei, M. Nishioka, and M. Shin, 1990,J. Biochem.107, 105–110). These three gpGSTs were quantitatively retained on anS-hexyl-GSH affinity column and separated as homogeneous proteins by chromatofocusing. Subunits of the heterodimers were inseparable on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but could be completely separated by reverse-phase partition high-performance liquid chromatography. A molecular cloning study demonstrated that the gpGST subunit M2 consisted of 217 amino acid residues with a calculated molecular mass of 25,562 and shared 84% identity in overall amino acid sequence with gpGSTM1-1. N-terminal amino acid sequences of peptides from the gpGST subunit M3* with a blocked N-terminus strongly suggested that it should belong to the Mu class. Western blot analysis using antisera raised against purified rat (r) GSTsA1-2 (Alpha), M1-1, P1-1 (Pi), and T2-2 (Theta) indicated that gpGSTsM1-1 and M1-3* cross-reacted only with anti-rGSTM1 antibody. However, gpGSTM1-2 cross-reacted intensely to almostthe same extent with antibodies to both rGSTsM1-1and T2-2. A homodimeric gpGSTM2-2, artificially constructed from native gpGSTM1-2 by treatment with guanidine hydrochloride followed by dialysis, intensely cross-reacted with antibodies to both the rat Mu- and Theta-class GSTs. Thus, the gpGST subunit M2 provided the first evidence for the double immuno-cross-reaction of a GST with polyclonal antibodies to two different classes of GSTs.